This application relates to methods and compositions for synthesizing oligosaccharides, and to the oligosaccharide products which can be obtained using such methods and compositions.
Oligosaccharide are compounds with considerable potential both as therapeutics and as reagents for clinical assays. The very nature of the saccharide subunits, however, makes the synthesis of many oligosaccharide of potential interest a daunting task because of the many possibilities for formation of positional isomers in which different substituent groups on the sugars become involved in bond formation and potential for the formation of different anomeric forms. Because of these factors, chemical synthesis of most oligosaccharides while possible is not generally feasible on a commercial scale because of poor yields of the desired product.
An alternative to chemical synthesis of oligosaccharides is enzymatic synthesis. In particular, enzymatic synthesis using glycosyl transferases, glycosidases or combinations thereof has been considered as a possible approach to the synthesis of oligosaccharides.
Glycosyl transferases catalyze the reaction
Glycosyl transferases can be very effective for producing specific products with good stereochemical and regiochemical control, if a transferase with the desired specificity is available. The enzymes can be expensive and hard to handle since they are often membrane-associated and unstable, however, and the required nucleotide sugar substrates can be quite expensive. Furthermore, glycosyl transferases possessing the desired specificity to make many interesting oligosaccharides are not available.
Glycosidases catalyze the reaction
and synthesize oligosaccharides when the reaction is run in reverse from the normal direction. In addition, oligosaccharide synthesis can be achieved by adding a second sugar to the reaction mixture which competes with water and reacts in its place with the first sugar in a transglycosylation reaction. Glycosidases are generally available and easy to handle and have the potential to make many different products using inexpensive substrates. Unfortunately, it is difficult to control the reverse hydrolysis reaction which leads to poor product yields. In addition, while the stereochemical control (i.e., the formation of only one anomer) is generally good, it is hard to predict or control the regiochemistry (i.e., the formation of 1–2 vs 1–3 vs 1–4 vs 1–6 bonds).
To realize the potential of enzymatic oligosaccharide synthesis, there is therefore a need for a synthetic approach which avoids the drawbacks of the known techniques. It is an object of this invention to provide such a technique which permits the synthesis of a wide variety of oligosaccharides in good yield, and to provide enzymes suitable for practicing these techniques.